(2000) The biochemical characterization of the DNA binding activity of pKi-67. granular components of the nucleolus. Finally, spatial analyses by electron tomography showed that pKi-67 forms cords 250C300 nm in diameter, which are themselves composed of 30C50-nm-thick fibers. These detailed comparative in situ analyses strongly suggest the involvement of pKi-67 in the higher-order organization of perinucleolar chromatin. Keywords: confocal microscopy, electron tomography, heterochromatin, Ki-67 antigen, nucleolus Almost two decades ago, a mouse monoclonal antibody (MAb), designated Ki-67, was reported to react with a nuclear antigen expressed only in proliferating cells (Gerdes et al. 1983). The Ki-67 antigen (pKi-67) is detected in nucleoli of R935788 (Fostamatinib disodium, R788) cycling cells (G1, S, G2) and at the periphery of mitotic chromosomes (van Dierendonck et al. 1989; Traut et al. 2002). In contrast, pKi-67 has never been detected in resting cells (G0) (Gerdes et al. 1984). Since Rabbit Polyclonal to REN that time, pKi-67 has been widely used as a prognostic indicator for estimation of the growth fraction of clinical samples from human neoplasms (Scholzen and Gerdes 2000). However, despite its usefulness for assessment of cell proliferation, little is known about the role of pKi-67 in vivo (Endl and Gerdes 2000). As revealed by Western blotting, pKi-67 is a large protein consisting of two main variants. These isoforms (with theoretical molecular masses of 320 and 359 kD) are obtained by alternative splicing of a mRNA precursor encoded by a unique gene (Gerdes et al. 1991; Duchrow et al. 1994). Analysis of the pKi-67 primary sequence has not revealed any significant homology to other known sequences. However, several putative nuclear targeting sequences have been identified, as well as more than a hundred potential phosphorylation sites (Schlter et al. 1993). In addition, several striking features have been determined. Both variants of the protein contain sixteen repetitive elements (Ki repeats), each of which includes a 66-bp motif, the Ki motif, which is highly conserved (Schlter et al. 1993). Moreover, a forkhead-associated (FHA) domain has been found in the N-terminal portion of pKi-67 (Sueishi et al. 2000). This domain, believed to be a modular phosphopeptide recognition motif that might mediate protein-protein interactions (Henckel et al. 1999; Li et al. 2000), is shared by several proteins involved in cell cycle regulation (Hofmann and Bucher 1995). This finding can be related to previous data, which revealed the role played by pKi-67 in cell cycle progression. Indeed, it has been reported that Ki-67 specific antisense oligonucleotides prevent incorporation of [3H]-thymidine (Schlter et al. 1993) and that microinjection of antibodies directed against the murine homologue of pKi-67 delays cell cycle progression (Starborg et al. 1996). Many data suggest that pKi-67 might be involved in the organization of chromatin higher-order structure (Takagi et al. 1999; MacCallum and Hall 2000). This R935788 (Fostamatinib disodium, R788) hypothesis is indirectly supported by other evidence. Ki-67 immunolabeling disappears after digestion with DNase I but not after RNase treatment (Sasaki et al. 1987). Moreover, Ki-67 antibodies display a stronger affinity when pKi-67 is bound to DNA (Lopez R935788 (Fostamatinib disodium, R788) et al. 1994). In addition, an increase of pKi-67 follows the increase of DNA during S-phase, whereas the global protein content decreases. Finally, recent biochemical data obtained by subcellular fractionation have confirmed that pKi-67 is a chromatin-associated protein, which probably resides in densely packed regions such as heterochromatin (Kreitz et al. 2000). Although many data support an involvement of pKi-67 in chromatin organization, some contradictory studies have localized pKi-67 mainly within the nucleolus, R935788 (Fostamatinib disodium, R788) in close association with the nucleolar components that are directly involved in rRNA elongation and maturation (Verheijen et al. 1989; Kill 1996; MacCallum and Hall 2000) or in association with a new nucleolar protein.